Human ASGR1 ORF clone (NM_001671) (Cat. No.: V029283)
Note: This is a 9093 bp third-generation lentiviral expression vector designed for dual expression. It drives constitutive expression of the human ASGR1 gene (tagged with 3xFLAG) via the CMV promoter. Additionally, it co-expresses mCherry and the PuroR selection marker under the EF-1α core promoter and an IRES element, respectively. The vector includes all essential HIV-1 packaging components (5' LTR, RRE, cPPT/CTS, WPRE) to ensure efficient viral production and stable transduction in mammalian cells.
- Name:
- pLV3-CMV-ASGR1(human)-3xFLAG-EF1a-mCherry-Puro
- Accession ID:
- NM_001671.5
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9093 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro;mCherry
- Promoter:
- CMV
- Fusion Tag:
- 3xFLAG
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
- Expression Method:
- Constiutive, Stable / Transient
- Gene Synonyms:
- ASGPR; ASGPR1; CLEC4H1; HL-1
- Transcript Definition:
- Homo sapiens asialoglycoprotein receptor 1 (ASGR1), transcript variant 1, mRNA
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
Human ASGR1 ORF clone (NM_001671) (Cat. No.: V029283) Sequence
LOCUS V029283 9093 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V029283
VERSION V029283
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
LTR 234..414
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
/label="5' LTR (truncated)"
misc_feature 458..583
/note="packaging signal of human immunodeficiency virus
type 1"
/label="HIV-1 Ψ"
misc_feature 1076..1309
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
/label="RRE"
CDS 1493..1537
/codon_start=1
/note="recognized by the 2H10 single-chain llama nanobody"
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/transl_table=1
/translation="KNEQELLELDKWASL"
/label="gp41 peptide"
misc_feature 1804..1921
/note="central polypurine tract and central termination
sequence of HIV-1"
/label="cPPT/CTS"
promoter 2011..2214
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 2303..3175
/label="ASGR1(NM_001671)"
/note="ASGR1(NM_001671)"
/gene="ASGR1"
CDS 3182..3247
/codon_start=1
/product="three tandem FLAG® epitope tags, followed by an
enterokinase cleavage site"
/transl_table=1
/translation="DYKDHDGDYKDHDIDYKDDDDK"
/label="3xFLAG"
promoter 3291..3502
/note="core promoter for human elongation factor
EF-1α"
/label="EF-1α core promoter"
LTR 3515..3783
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
/label="5' LTR (truncated)"
CDS 3818..4525
/codon_start=1
/note="mammalian codon-optimized"
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/transl_table=1
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
/label="mCherry"
CDS 4535..4588
/codon_start=1
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/product="2A peptide from Thosea asigna virus capsid
protein"
/protein_id=""
/transl_table=1
/translation="EGRGSLLTCGDVEENPGP"
/label="T2A"
CDS 4589..5188
/codon_start=1
/gene="pac from Streptomyces alboniger"
/note="confers resistance to puromycin"
/product="puromycin N-acetyltransferase"
/transl_table=1
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
/label="PuroR"
misc_feature 5189..5776
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
/label="WPRE"
LTR 5850..6083
/note="self-inactivating 3' long terminal repeat (LTR)
from HIV-1"
/label="3' LTR (ΔU3)"
polyA_signal 6155..6276
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
rep_origin 6295..6430
/note="SV40 origin of replication"
/label="SV40 ori"
primer_bind complement(6468..6484)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
protein_bind 6492..6508
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG)."
/label="lac operator"
promoter complement(6516..6546)
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 6561..6582
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
rep_origin complement(6870..7458)
/direction=LEFT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
CDS complement(7629..8489)
/codon_start=1
/gene="bla"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/product="β-lactamase"
/transl_table=1
/translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
SLIKHW*"
/label="AmpR"
promoter complement(8490..8594)
/gene="bla"
/label="AmpR promoter"
primer_bind 9068..9084
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 fwd"